Lactic acid bacteria and their use in swine direct-fed microbials

ABSTRACT

TRFs useful for identifying strains of interest are provided. A method of identifying one or more strain that can be used as a direct-fed microbial is also provided. One or more strain identified by the method is additionally provided. A method is also provided for administering to an animal an effective amount of the one or more strain. Additionally provided is an isolated strain chosen from at least one of  Lactobacillus acidophilus  strain P1B c6 (NRRL B-50103),  Lactobacillus salivarius  strain o246e 33w (NRRL B-50102),  Pediococcus acidilactici  strain o246e 42 (NRRL B-50171), and  Pediococcus acidilactici  strain P1J e3 (NRRL B-50101). An isolated strain having all of the identifying characteristics of one of the strains listed above is also provided. One or more strain can be administered as a direct-fed microbial to an animal. Methods of preparing a direct-fed microbial are also provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional patent application of U.S. patentapplication Ser. No. 12/685,979 filed Jan. 12, 2010, which claimspriority under 35 U.S.C. §119(e) to U.S. Provisional Patent ApplicationNo. 61/143,990 filed Jan. 12, 2009; the entirety of each application isincorporated by reference herein.

BIBLIOGRAPHY

Complete bibliographic citations of the references referred to herein bythe first author's last name and year of publication in parentheses canbe found in the Bibliography section, immediately preceding the claims.

FIELD OF THE INVENTION

The invention relates to biological methods and products useful inagriculture. More particularly, though not exclusively, the presentinvention relates to lactic acid bacteria, methods of administeringlactic acid bacteria to animals, such as pigs, and methods of makinglactic acid bacteria.

DESCRIPTION OF THE RELATED ART

The swine industry has implemented the practice of early weaning forefficient and economical pig production (Wilson, 1995). The obviousconsequence of weaning is the abrupt change in diet from sow's milk tosolid feed and a change in the pigs' social environment (McCracken etal., 1995). There is reduced feed intake during the first week andassociated adverse changes in the animal's gut anatomy and physiologysuch as villus atrophy, deeper crypts, and infiltration of the villustip by immature enterocytes. Villus atrophy means that there is lessabsorptive area available for nutrient uptake and deeper cryptsrepresent a large tissue turnover (Cera et al., 1988). Previous researchhas reported that villus height-to-crypt depth ratios are altered inresponse to different weaning environments and populations of residentgastrointestinal microflora populations (Tang et al., 1999). Adisruption of the intestinal microflora often accompanies abruptweaning, adversely impacting stability of the gastrointestinal tract,and this disruption may be the impetus for the alterations in feedintake and gut anatomy that lead to poor growth and compromised healthduring the post-weaning period (Dritz et al., 1996). Combined, theseconditions have dramatic detrimental effects on piglet growth andhealth, negatively impacting the development of a mature digestivesystem and effective immune defense, with ramifications on the pig'sgrowth performance through later production cycles.

Several approaches have evolved in modern swine production to addressthese issues while still capitalizing on the efficiency and economicbenefits of early weaning, including management and diet changes.Early-weaning at an age of less than 21 days followed by removal of pigsto a second isolated site is commonly referred to as segregated earlyweaning (SEW). This approach reduces the incidence of a number ofpathogens, thus reducing immunological stress, resulting in improvedgrowth and higher efficiency of feed utilization (Fangman et al., 1997).This strategy has been successful in reducing the number of pathogens,but has not been successful in eliminating all pathogens. The premise isthat pigs are removed from the sow while their immunity, as aconsequence of maternal antibodies, is still high. This maternallyderived passive immunity will prevent vertical transfer of indigenouspathogens. Whereas the gastrointestinal disruptions from abrupt weaningare not eliminated by SEW, the problems seem to be much less inprevalence and severity when pigs are weaned early and to an isolatedfacility off the farm site. This is likely a consequence of lesspathogenic challenge to the pigs at weaning when their innate defensesare compromised.

Diet formulations utilizing good quality protein sources and additivesto ease the weaning transition are an additional approach to protectpiglet health. Two of the most prevalent options used are the inclusionof spray-dried plasma protein and in-feed antibiotics. The benefits ofantibiotic supplementation to swine diets has been well documented, withthe greatest improvements in responses occurring in response toantibiotic addition to weanling pigs compared to pigs in later growingstages (reviewed by Cromwell, 2001). The performance-enhancing benefitsof spray-dried animal plasma (SDAP) are extensively documented as wellwithin the scientific literature (reviewed by van Dijk et al., 2001a),and SDAP is particularly highly regarded as an ingredient in weanlingpig starter diets. The addition of SDAP consistently results in improvedbody weight gain and feed intake, as well as reduction in post-weaningdiarrhea, particularly during the one- to two-week time period followingweaning (Coffey and Cromwell, 1995; van Dijk et al, 2001a; Bikker etal., 2004). Proposed mechanisms reported previously in the scientificliterature include: improved diet palatability, improved digestibilityof nutrients, the presence of beneficial growth factors, pathogenbinding/blocking glycoproteins, neutralization of toxins; the presenceof immunoglobulins, immunomodulation, improved intestinal morphology,and changes in gut microbial ecology (Bosi et al., 2001, 2004; Hammer etal., 2004; Mouricout et al., 1990; Nollet et al., 1999; Perez-Bosque etal., 2004; Roche et al., 2000; Torrallardona et al., 2003; van Dijk etal., 2001a; van Dijk et al., 2002b).

The effects of these and other dietary additives lend support to theconcept that the effects of luminal nutrients and additives have less ofa direct impact on the pig and instead act by mediating microbial shiftsin response to exogenous nutrient availability (Gaskins, 2001). This hasled to great interest in the use of direct-fed microbial (DFM) additivesto aid the weaning transition by preventing the disruption of thegastrointestinal ecosystem that paves the way for pathogen invasion,thereby promoting growth and maintaining health in the young pig.Supplementation of Bacillus cultures has been reported to improve growthperformance in weanling pigs (Yang et al., 2003; Kyriakis et al., 1999;Adami et al., 1997) by affording the pig protection against pathogenicchallenges. Whereas most of the DFM additives commonly fed to swine areBacillus-based, supplementation with lactobacilli also provideperformance benefits to the young pig. Salmonella-challenged pigssupplemented with a five-strain combination of lactobacilli had improvedweight gain, less severity of symptoms, and reduced fecal shedding ofSalmonella compared to unsupplemented pigs that were challenged (Caseyet al., 2007). Also, Lactobacillus brevis supplemented to the neonatalpig resulted in goblet cell maturation in the small intestine anddramatic improvements in body weight gain following weaning, that seemto be a consequence of control of inflammatory signals in the gutthrough toll-like receptor signaling (Davis et al., 2006, 2007). Withthe stigma of antibiotic resistance associated with growth promotinglevels of antimicrobials fed to livestock and the use of animalbyproducts like plasma protein with recent concerns regardingTransmissible Spongiform Encephalitis, supplementation of beneficialbacteria to provide some of the same benefits has gained popularity inthe livestock industries.

Although there is a paucity of scientific support for their efficacy andlimited understanding of their mode of action, supplementation withprobiotic products has become increasingly popular in both human andagriculture sectors. Historically, probiotics originated from theconcept that individuals that consumed large quantities of fermenteddairy products, such as yogurt and cheese, were especially long-lived(Tannock, 2004). The majority of probiotic organisms are selectedbecause they are easily propagated and readily available from foodfermentation processes, with very little scientific support guidingtheir selection (Fuller, 1997). Therefore, bacteria used for DFMproducts for livestock species are not usually selected to providebacterial organisms that would be ideally suited for the challengespresent in livestock production systems.

What is needed are bacterial strains that are useful in pigs and otheranimals. Methods of making and using bacterial strains are also needed.In addition, DNA sequences for identifying these strains and methods ofidentifying strains with the DNA sequences are also needed.

SUMMARY OF THE INVENTION

The invention, which is defined by the claims set out at the end of thisdisclosure, is intended to solve at least some of the problems notedabove.

A DNA sequence is provided. The DNA sequence includes a portion of the16S rRNA gene coding region of a lactic acid bacteria, the 5′ end ofwhich includes the sequence of 5′ AGAGTTTGATYMTGGCTCAG 3′, the 3′ end ofwhich includes a restriction enzyme recognition site for one of Bfa I,Hae III, and Msp I. When the restriction enzyme recognition site is BfaI, the DNA sequence has a length of about 99 base pairs to about 103base pairs, about 268 base pairs to about 273 base pairs, about 260 basepairs to about 265 base pairs, about 279 base pairs to about 284 basepairs, about 278 base pairs to about 282 base pairs, or about 273 basepairs to about 277 base pairs. When the restriction enzyme recognitionsite is Hae III, the DNA sequence has a length of about 329 base pairsto about 334 base pairs, about 352 base pairs to about 357 base pairs,about 277 base pairs to about 282 base pairs, or about 335 base pairs toabout 339 base pairs. When the restriction enzyme recognition site isMsp I, the DNA sequence having a length of about 188 base pairs to about192 base pairs.

A method of identifying one or more strain that can be used as adirect-fed microbial is also provided. In the method, DNA is isolatedfrom bacteria from the gastrointestinal tract of an animal. The DNA isamplified. The amplified DNA is analyzed with T-RFLP to generate TRFdata. The TRF data is correlated to a characteristic of interest.Strains of interest are identified from the correlations. The presenceof the TRF in the strain is confirmed.

One or more strain identified by the method is additionally provided. Amethod is also provided for administering to an animal an effectiveamount of the one or more strain.

In addition, an isolated Pediococcus acidilactici strain P1J e3 (NRRLB-50101) is provided, along with a combination including the isolatedPediococcus acidilactici strain P1J e3 (NRRL B-50101) and an isolatedLactobacillus salivarius strain o246e 33w (NRRL B-50102).

Additionally provided is an isolated strain chosen from at least one ofLactobacillus acidophilus strain P1B c6 (NRRL B-50103), Lactobacillussalivarius strain o246e 33w (NRRL B-50102), Pediococcus acidilacticistrain o246e 42 (NRRL B-50171), and Pediococcus acidilactici strain P1Je3 (NRRL B-50101). An isolated strain having all of the identifyingcharacteristics of one of the strains listed above is also provided.

Additional methods are provided. In one, an effective amount of at leastone strain chosen from Lactobacillus acidophilus strain PIB c6 (NRRLB-50103), Lactobacillus salivarius strain o246e 33w (NRRL B-50102),Pediococcus acidilactici strain o246e 42 (NRRL B-50171), and Pediococcusacidilactici strain PH e3 (NRRL B-50101) is administered to an animal.

Another method is a method of preparing a direct-fed microbial. In it,in a liquid nutrient broth, at least one strain chosen fromLactobacillus acidophilus strain P1B c6 (NRRL B-50103), Lactobacillussalivarius strain o246e 33w (NRRL B-50102), Pediococcus acidilacticistrain o246e 42 (NRRL B-50171), and Pediococcus acidilactici strain P1Je3 (NRRL B-50101) is grown. The strain is separated from the liquidnutrient broth to form the direct-fed microbial.

BRIEF DESCRIPTION OF THE DRAWINGS

Exemplary embodiments of the invention are illustrated in theaccompanying drawings.

FIG. 1 is a graph displaying the concentration of lymphocytes on day 1,3, 10, and 24 post-weaning (or 20, 22, 29 and 43 d of age) isolated fromthe peripheral blood of nursery pigs farrowed in conventional indoorfacilities compared to those farrowed in outdoor facilities(Treatment×Day interaction, P<0.01; a,b, Means within each day withdiffering letters are different (P<0.05).

FIG. 2A-2B are two halves of a Dendrogram displaying the similaritybetween the nine candidate DFM strains as determined by RAPD primer 1and 4 fingerprints. The TRF banding patterns of the nine strains for therestriction endonucleases Bfa I, Hae III, and Msp I are also shown.

FIG. 3 is a graph displaying initial and final coefficient of variationof body weight within litter resulting from the administration ofprobiotic combinations to sows during the lactation period(Treatment×time interaction, P=0.06; SE=1.22). PA=Pediococcusacidilactici P1J e3; LA=Lactobacillus acidophilus P1B c6;LS=Lactobacillus salivarius o246e 33w.

Before explaining embodiments of the invention in detail, it is to beunderstood that the invention is not limited in its application to thedetails of construction and the arrangement of the components set forthin the following description or illustrated in the drawings. Theinvention is capable of other embodiments or being practiced or carriedout in various ways. Also, it is to be understood that the phraseologyand terminology employed herein is for the purpose of description andshould not be regarded as limiting.

DETAILED DESCRIPTION

In accordance with the present invention, there may be employedconventional molecular biology and microbiology within the skill of theart. Such techniques are explained fully in the literature. See, e.g.,Sambrook, Fritsch & Maniatis, Molecular Cloning: A Laboratory Manual,Third Edition (2001) Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y.

Described herein are novel lactic acid bacteria strains and combinationsthereof. The strains, alone or in combination, can be administered toanimals, such as pigs. When fed to pigs, the strains provide manybenefits. When the strains are fed to pregnant sows or gilts or tolactating sows, benefits are seen in their offspring. The strains alsoprovide benefits when fed to pigs of other ages, such as nursery pigsand grow/finish pigs.

A method of identifying one or more strain that can be used as adirect-fed microbial (DFM) is also described herein. In the method, DNAis isolated from bacteria from gastrointestinal track of an animal. TheDNA is amplified. The amplified DNA is analyzed with TerminalRestriction Fragment Length Polymorphism (T-RFLP) to generate terminalrestriction fragment (TRF) data. The TRF data is correlated to acharacteristic of interest. Strains of interest are identified from thecorrelations. The number of strains is reduced with RAPD PCR analysis ofthe strains or any other suitable method. The presence of the TRF in thestrain is confirmed.

Terminal Restriction Fragments (TRFs):

Described herein are TRFs useful for identifying strains of interest.Those TRFs include, but are not limited to, a DNA sequence that includesa portion of the 16S rRNA gene coding region of a lactic acid bacterium.The 5′ end of the DNA sequence includes the sequence of 5′AGAGTTTGATYMTGGCTCAG 3′, and the 3′ end includes a restriction enzymerecognition site for one of Bfa I, Hae III, and Msp I. When therestriction enzyme recognition site is Bfa I, the DNA sequence has alength of about 99 base pairs to about 103 base pairs, about 268 basepairs to about 273 base pairs, about 260 base pairs to about 265 basepairs, about 279 base pairs to about 284 base pairs, about 278 basepairs to about 282 base pairs, or about 273 base pairs to about 277 basepairs. When the restriction enzyme recognition site is Hae III, the DNAsequence has a length of about 329 base pairs to about 334 base pairs,about 352 base pairs to about 357 base pairs, about 277 base pairs toabout 282 base pairs, or about 335 base pairs to about 339 base pairs.When the restriction enzyme recognition site is Msp I, the DNA sequencehas a length of about 188 base pairs to about 192 base pairs.

Lactic Acid Bacteria:

Also described herein are strains of lactic acid bacteria that wereidentified using terminal restriction fragments (TRF): Lactobacillusacidophilus PIB c6, L. salivarius o246e 33w, Pediococcus acidilacticio246e 42, and P. acidilactici P1J e3. These strains were deposited atthe Agricultural Research Service Culture Collection (NRRL), 1815 NorthUniversity Street, Peoria, Ill., 61604 as follows. Lactobacillusacidophilus P1B c6 was deposited on Jan. 18, 2008 and received accessionnumber NRRL B-50103, L. salivarius o246e 33w was deposited on Jan. 18,2008 and received accession number NRRL B-50102, Pediococcusacidilactici o246e 42 was deposited on Aug. 29, 2008 and receivedaccession number NRRL B-50171, and P. acidilactici P1J e3 was depositedon Jan. 18, 2008 and received accession number NRRL B-50101. Additionalstrains identified with the TRFs described herein are also within thescope of the invention.

A lactic acid bacteria strain, Lactobacillus brevis 1E1, can be usedwith one or more of the strains listed above. Strain 1E-1 (also writtenas strain 1E1) was isolated from the intestinal tract of a healthy,weaned pig. Strain 1E-1 is available from the microorganism collectionof the American Type Culture Collection, 10801 University Blvd.,Manassas, Va. 20110, under accession number PTA-6509, and was depositedon Jan. 12, 2005. All of the deposits were made under the provisions ofthe Budapest Treaty on the International Recognition of the Deposit ofMicroorganisms for the Purposes of Patent Procedure.

Strains having all the identifying characteristics (as provided below)of the strains listed above are also considered within the scope of theinvention.

In brief, strains of lactic acid bacteria were identified using TRFs asfollows with further details of this below in the Examples section. Pigswere separated into two groups with different conditions to producedifferences in performance and immune measurements, e.g., specificimmune subpopulations of immune cells. As used herein, “performance”refers to the growth of an animal measured by the following parameters:average daily weight gain, total weight gain, feed conversion, whichincludes both feed:gain and gain:feed, feed efficiency, mortality, andfeed intake. “An improvement in performance” as used herein, means animprovement in at least one of the parameters listed above under theperformance definition.

From the group of pigs having measurements for performance and immunecharacteristics, TRFs were identified from the lactic acid bacteria fromtheir gastrointestinal tracks. These TRFs were present in the group ofpigs having beneficial measurements and were absent in the group of pigslacking beneficial measurements. The number of potentially usefulstrains was narrowed using RAPD PCR analysis. The existence of the TRFsin the narrowed group of strains was confirmed. Because those TRFs werefound in the group of pigs having better measurements, the inventorsbelieved that those strains should positively affect performance and/orimmune modulation. When fed to animals, four strains of lactic acidbacteria were found to positively affect performance.

The strains can be fed alone or in combination. In one exemplaryembodiment, strains Lactobacillus acidophilus PIB c6, Lactobacillussalivarius o246e 33w, and Pediococcus acidilactici P1J e3 are combinedand administered to animals such as lactating sows and/or to piglets. Inanother exemplary embodiment, strains Pediococcus acidilactici P1J e3and Lactobacillus salivarius o246e 33w are combined and administered. Inanother exemplary embodiment, Pediococcus acidilactici P1J e3 alone isadministered to animals such as lactating sows and/or to piglets.

Direct-Fed Microbials:

A direct-fed microbial (DFM), which is used interchangeably throughoutthis disclosure with “probiotic,” includes one or more strain of lacticacid bacteria listed above. One or more carrier or other ingredients canbe added to the DFM. The DFM may be presented in various physical forms,for example, as a top dress, as a water soluble concentrate for use as aliquid drench or to be added to a milk replacer, gelatin capsule, orgels. In one embodiment of the top dress form, freeze-dried lactic acidbacteria fermentation product is added to a carrier, such as whey,maltodextrin, sucrose, dextrose, limestone (calcium carbonate), ricehulls, yeast culture, dried starch, and sodium silico aluminate. In oneembodiment of the water soluble concentrate for a liquid drench or milkreplacer supplement, freeze-dried lactic acid bacteria fermentationproduct is added to a water soluble carrier, such as whey, maltodextrin,sucrose, dextrose, dried starch, sodium silico aluminate, and a liquidis added to form the drench or the supplement is added to milk or a milkreplacer. In one embodiment of the gelatin capsule form, freeze-driedlactic acid bacteria fermentation product is added to a carrier, such aswhey, maltodextrin, sugar, limestone (calcium carbonate), rice hulls,yeast culture dried starch, and/or sodium silico aluminate. In oneembodiment, the lactic acid bacteria and carrier are enclosed in adegradable gelatin capsule. In one embodiment of the gels form,freeze-dried lactic acid fermentation product is added to a carrier,such as vegetable oil, sucrose, silicon dioxide, polysorbate 80,propylene glycol, butylated hydroxyanisole, citric acid, ethoxyquin, andartificial coloring to form the gel.

To obtain the lactic acid bacteria and to form a DFM, the lactic acidbacteria can be fermented to an appropriate level. In a non-limitingexample, that level is between about a 1×10⁹ CFU/ml level to about a1×10¹⁰ CFU/ml level. These lactic acid bacteria can be grown in de Man,Rogosa and Sharpe (MRS) broth at 37° C. for 24 hours. The bacteria canbe harvested by centrifugation, and the supernatant removed.

The pelleted lactic acid bacteria can then be fed as a DFM to an animal,such as a pig. In some embodiments, the DFM is fed to a sow, a gilt, apre-weaned piglet, a post-weaned piglet, or a pig of any age. The lacticacid bacteria can be fed to a sow during the lactation period, althoughthe lactic acid bacteria can be fed for different durations and atdifferent times. When fed to a gilt or sow, the strains are transferredto piglets borne to the gilt or sow. It is believed that this isaccomplished via the fecal-oral route and/or via other routes. In oneembodiment, pelleted lactic acid bacteria are freeze-dried for directfeeding to the animal. In at least some embodiments, lactic acidbacteria are added to animal feed.

When fed to an animal, lactic acid bacteria become established in itsgastrointestinal tract. About 1×10⁸ CFU/animal/day to about 5×10¹⁰CFU/animal/day of lactic acid bacteria can be fed regardless of whetherthe total CFU is derived from one organism or a combination oforganisms. In an exemplary embodiment, 1×10⁹ total CFU/animal/day oflactic acid bacteria is fed regardless of whether the total CFU isderived from one organism or a combination of organisms. In oneembodiment, equal amounts of each strain are used. In anotherembodiment, unequal amounts are used.

EXAMPLES

The following Examples are provided for illustrative purposes only. TheExamples are included herein solely to aid in a more completeunderstanding of the presently described invention. The Examples do notlimit the scope of the invention described or claimed herein in anyfashion.

Example 1 Conventional versus Segregated Early Weaning GrowthPerformance Model

A model to provide separation in growth performance was establishedusing pigs reared in off-site segregated early weaning managementconditions compared to pigs reared conventionally on the same farm sitein which birthing and rearing by the dam occurred. Eighty-eightcrossbred barrows and gilts from 11 litters were weaned at 19 days ofage. One group of 44 pigs were moved to the segregated nursery 12 kmaway from the sow herd, whereas the remaining pigs were moved to anursery facility located at the pre-weaning location. Pigs were allottedinto 16 pens at each facility and body weight and feed disappearancewere measured on day 11, 18, and 25 after weaning to determine averagedaily gain (ADG), average daily feed intake (ADFI), and gain:feed. Fourpigs from each facility were selected for sampling on day 1, 3, 11, and25 after weaning, in which a blood sample was obtained via vena cavapuncture for cell isolation and pigs were humanely euthanized to obtaingastrointestinal tract tissues for microbial analyses and immune cellisolation. Table 1 below illustrates the improved (P<0.10) average dailygain and average daily feed intake responses observed for pigs reared ina segregated early weaning management system compared to conventionallyreared pigs, as well as the greater (P<0.01) pig body weight observedfor segregated early weaned pigs at the end of the study (25 days afterweaning).

TABLE 1 Growth performance responses of weanling pigs reared in anoff-site segregated early weaning (SEW) management system compared topigs reared conventionally on-site (CONV). CONV SEW SEM¹ P = d 0 to 11post-weaning ADG, g 217 388 12.37 0.001 ADFI, g 181 410 14.36 0.001Gain:feed 1.47 0.94 0.21 0.088 d 11 to 25 post-weaning ADG, g 466 47021.30 0.904 ADFI, g 587 591 29.80 0.927 Gain:feed 0.83 0.86 0.06 0.737 d0 to 25 post-weaning ADG, g 383 442 23.66 0.081 ADFI, g 452 531 30.870.075 Gain:feed 0.85 0.83 0.09 0.585 Pig weight, kg Initial 5.94 5.870.07 0.479 d 11 8.10 9.51 0.27 0.001 d 18 11.95 12.71 0.28 0.064 d 2515.85 17.38 0.35 0.003 ¹standard error of the mean (SEM)¹ standard error of the mean (SEM)

Example 2 Outdoor vs Conventional Rearing Growth Performance Model

A second model was established to provide a more robust separation ingrowth performance between young pigs, in which pigs were either rearedin conventional confinement farrowing facilities or farrowed in anoutdoor management system. One hundred forty-four pigs were identifiedat each facility for the experiment. Conventionally reared pigs werelocated in Indiana, whereas the outdoor reared pigs were located inColorado. Pigs at both facilities were of similar genetic background(PIC C-22×PIC 280). Six pigs from each facility, i.e., outdoor andindoor, were randomly selected to be sacrificed at each time interval ofsix and 14 days of age and 24 hrs prior to weaning (18 days of age) tomeasure gastrointestinal microbial populations and immune celldevelopment during the pre-weaning period. One hundred and twenty-sixpigs from each facility were weaned at 19 days of age and moved to anoff-site nursery facility located in Arkansas. Upon arrival, pigs fromeach group were placed in separate rooms within the same facility tomonitor growth performance during the post-weaning period while keepingthe groups segregated to prevent exposure to the microflora between thetwo groups. Pig body weight and feed disappearance was determined at theend of each dietary phase, defined as Phase 1 (day 0 to 11post-weaning), Phase 2 (day 11 to 25 post-weaning) and Phase 3 (day 25to 39 post-weaning). Six pigs from each group were selected for samplingon day 1, 3, 7, 10, and 24 post-weaning, in which a blood sample wasobtained via vena cava puncture for cell isolation and pigs werehumanely euthanized to obtain gastrointestinal tract tissues formicrobial analyses and immune cell isolation. Pigs farrowed inconventional sow facilities had numerically greater (5.18 vs. 5.74

±0.28; P=0.16) initial body weight at weaning compared to pigs reared inoutdoor facilities. However, pigs previously reared outdoors had greater(P<0.01) average daily gain, average daily feed intake, and body weightat the end of each phase than pigs farrowed in conventional confinementfacilities (see Table 2 below).

TABLE 2 Average daily gain (ADG), average daily feed intake (ADFI), andgain:feed (G:F), of nursery pigs farrowed by sows housed inconventional, confinement facilities and sows housed in outdoor pasturefacilities (with initial body weight of pigs used as a covariate in theanalysis). Outdoor Conventional SEM² P= ADG, g Phase 1 242 183 7 <0.01Phase 2 515 411 14 <0.01 Phase 3 658 594 15 <0.01 Phase 1-3 486 408 10<0.01 ADFI, g Phase 1 338 246 10 <0.01 Phase 2 727 581 12 <0.01 Phase 31059 929 14 <0.01 Phase 1-3 720 596 10 <0.01 Gain:feed Phase 1 0.7240.751 0.022 0.40 Phase 2 0.750 0.743 0.009 0.62 Phase 3 0.661 0.6720.006 0.17 Phase 1-3 0.701 0.707 0.006 0.57 Body weight, kg Phase 1 8.147.47 0.08 <0.01 Phase 2 15.75 13.53 0.16 <0.01 Phase 3 25.58 22.27 0.26<0.01 ²standard error of the mean (SEM)

Example 3 Immune Measurements and Flow Cytometric Analysis

Immune cells were isolated from blood and gastrointestinal samples and abattery of immune measurements were obtained from both of the growthperformance models of Examples 1 and 2, including: differential whiteblood cell counts; peripheral blood mononuclear cell proliferation andcytokine proliferation; gastrointestinal morphology, goblet cellenumeration, and immunohistochemistry from jejunal tissue; and flowcytometric analysis on cells isolated from peripheral blood and jejunaltissue. Cell isolation methods and laboratory procedures have beenpreviously published in the scientific literature (Davis et al., 2004;Brown et al., 2006a; Brown et al., 2006b). Antibody panels used todefine immune cell subsets for immunohistochemistry and flow cytometricanalyses are displayed in Table 3 below.

TABLE 3 Monoclonal antibodies specific for swine leukocytes used todefine cell surface molecule expression and differential populations ofleukocytes derived from peripheral blood in immunohistochemistry andflow cytometric analyses. Monoclonal Cell type(s) expressingAntibodies^(a) Clone Isotype Specificy molecule CD2¹ PG168A** IgG₃ CD2Virtually all thymocytes, T lymphocytes, and NK cells CD3² PPT3* IgG₁κCD3_(ε) T lymphocytes CD4^(1,2) 74-12-4* IgG_(2b)κ CD4a T helperlymphocytes CD8^(1,2) 76-2-11* IgG_(2a)κ CD8 α chain Cytotoxic Tlymphocytes Monocyte/ 74-22-15 IgG_(2bκ) SWC3a Granular leukocytesGranulocyte¹ CD25 (IL-2R)^(1,2) PGBL25A** IgG¹ CD25 (IL-2 R)Interleukin-2 receptor; activated T and B lymphocytes MHC-II^(1,2)MSA3** IgG_(2a) MHC-II molecule Monocytes/macrophages, B and Tlymphocytes, etc. CD21² BB6- IgG₁κ CD21 Mature circulating B 11C9.6*(complement lymphocytes receptor 2) TCR1² PGBL22A** IgG₁ Po-TCR1-N4 (γδ)Antigen on T cells ^(a)Monoclonal antibodies are mouse anti-pig. ¹Usedfor immunohistochemistry analysis. ²Used for flow cytometric analysis,*Purchased from Southern Biotechnology Associates, Inc., Birmingham, AL.**Purchased from Veterinary Medical Research and Development, Inc.,Pullman, WA.

Differences in gastrointestinal development and health betweenconventionally and segregated early weaned pigs, as defined byintestinal morphology, goblet cell differentiation, and gastrointestinalimmune cell populations have been reported previously in the literature(Brown et al., 2006a). Immune development was also altered by the twopre-weaning management systems utilized in the conventionally rearedcompared to the outdoor rearing growth model. Specifically, lymphocytepopulations in the peripheral blood differed between the conventionaland outdoor management systems during the post-weaning period, in whichpigs reared previously outdoors had a lower (P<0.05) proportion oflymphocytes in the blood 3 days after weaning compared to conventionallyreared pigs but a greater (P<0.05) proportion 10 days after weaning(management system x day interaction, P<0.01; FIG. 1). Furthermore, flowcytometric analysis of peripheral blood mononuclear cells revealed pigsreared in conventional confinement facilities during the lactationperiod had a greater proportion of leukocytes expressing the activationmolecule, CD25, during the preweaning (40.90 vs. 26.95±4.99; P<0.05) andpost-weaning (34.78 vs 19.26±4.55; P<0.05) periods. These dataillustrate how the different rearing systems altered immune developmentboth during the period when pigs within each system were separated(pre-weaning) and when the two groups were brought into a similarpost-weaning management system. Differences were also evident ingastrointestinal development in which pigs reared in conventionalconfinement facilities had greater (P<0.01) villus height and lower(P<0.05) crypt depth within the duodenum before weaning compared tooutdoor reared pigs, whereas duodenal villus height, crypt depth, andarea were greater (P<0.01) in outdoor reared pigs compared toconventionally reared pigs after weaning (see Table 4 below). This isfurther evidenced by differences in immune cell development within thejejunum of the gastrointestinal tract. Examples of this include agreater (12.44 vs. 8.99±1.22; P<0.05) proportion of leukocytesexpressing the CD25 activation molecule prior to weaning, a greater(25.08 vs. 16.49±3.49; P<0.05) proportion of lymphocytes expressing CD8in pigs previously reared outdoors 24 days after weaning, and a greaterproportion of leukocytes expressing the antigen presenting molecule,major histocompatability complex-II (MHC-II) as evidenced byimmunohistochemistry analysis, during the pre-weaning (30.6 vs.18.4+4.4) and post-weaning (26.0 vs. 35.1+3.7) periods when pigs werereared in outdoor systems compared to conventional confinement systemsduring the lactation period.

TABLE 4 Gastrointestinal morphology (crypt depth, villus height, andvillus area) measurements from the duodenum, jejunum, and ileum of pigsfarrowed in outdoor and indoor management systems Pre-WeaningPost-Weaning Indoor Outdoor SEM³ P≦ Indoor Outdoor SEM⁴ P≦ DuodenumCrypt depth, μm 70.7 79.1 3.0 0.05 94.1 117.5 4.3 0.01 Villus height, μm450.8 390.0 15.5 0.01 332.7 388.0 12.41 0.01 Villus area, μm² 48,26742,100 2276 0.06 42,658 53,680 1968 0.01 Jejunum Crypt depth, μm 75.371.8 3.66 0.50 98.8 107.2 4.1 0.16 Villus height, μm 398.5 376.1 16.60.35 330.7 352.7 13.6 0.26 Villus area, μm² 35,394 34,804 2153 0.8537,917 43,980 2347 0.08 Ileum Crypt depth, μm 68.0 73.1 3.0 0.23 103.5114.9 4.9 0.11 Villus height, μm 288.4 422.2 106.1 0.38 508.0 308.8118.4 0.24 Villus area, μm² 32,529 27,993 1704 0.07 30,364 35,448 12600.01 ³standard error of the mean (SEM) ⁴standard error of the mean (SEM)³ standard error of the mean (SEM)⁴ standard error of the mean (SEM)

Example 4 Isolation and Identification of Gastrointestinal Bacteria

Tissue Processing.

Gastrointestinal sections including the pars esophagus, duodenum,jejunum and ileum were collected for bacterial cell isolation. Luminalmaterial was removed from each gut section by washing twice with 25 mLof sterile washing buffer (0.3 mM KH₂PO₄, 1 mM MgSO₄, and 0.05% cysteinehydrochloride, pH 7.0). Tract sections were cut transversely withsterile forceps and any remaining luminal material was again removedwith 25 mL sterile 0.1% Peptone dilution buffer. The gut section wasplaced in a sterile whirl-pak bag with 99 mL of sterile peptone dilutionbuffer and masticated in a stomacher for 30 seconds to releasecolonizing or mucus associated bacteria. The masticated solution waspoured into a sterile 250 mL centrifuge tube withholding the gutsection. Centrifugation at 13,170×g for 10 min. was performed on thebacterial cell-containing solution. Subsequently the supernatant wasdiscarded and 10 mL of sterile MRS+10% glycerol broth was added to thepellet, resuspended and frozen at −20° C. until subsequent DNAisolation.

DNA Isolation.

Frozen post-weaning gastrointestinal section samples (N=128) were thawedon ice prior to DNA isolation. Solutions were vortexed for 30 seconds toyield a heterogeneous sample and to break up aggregated cells or cellsthat may have become associated with globule material during freezing.Two mL of mixed cells were then filtered through sterile 1M Whatman milkfilter paper to remove globular material that interfered with the DNAisolation process. The DNA isolation process continued as follows: 0.5mL of cells were added to a sterile 15 mL conical tube and washed with15 mL of 50 mM Tris-HCl, 10 mM EDTA (T₅₀E₁₀) solution pH 7.5, followedby centrifugation at 2,485×g for 10 min. to remove PCR inhibitingsubstances. This washing and centrifugation step was repeated a secondtime. The pelleted cells were isolated following the directions of theRoche Genomic DNA Isolation Kit (Roche Diagnostics Corp., Indianapolis,Ind.), with slight modifications. All Phosphate Buffered Salinesolutions were replaced with T₅₀E₁₀ and a 100 mg/mL lysozyme solutiondissolved in T₅₀E₁₀ replaced the 5 mg/mL lysozyme solution recommended.After isolation, the DNA was quantified using a Picogreen dsDNAQuantitation kit (Molecular Probes, Eugene, Oreg.) and a TD-360Mini-Fluorometer (Turner Biosystems, Sunnyvale, Calif.).

PCR Amplification and T-RFLP Analysis.

Amplification reactions using 50 ng of DNA from each gut section sample(with the exception of the pars esophagus) were performed in triplicateto provide adequate quantity of amplified product and to reduce PCRvariation. The DNA from the pars esophagus section was oftenundetectable in this range, thus 2 μL from pars esophagus DNA isolationsamples were added to the PCR reaction. A 5′-tetrachlorofluoresceinlabeled 8F domain primer (5′ AGAGTTTGATYMTGGCTCAG 3′) and a 1406Runiversal primer (5′ ACGGGCGGTGTGTRC 3′) were used to amplify a largeportion of the 16S rRNA gene coding region (Baker, et al., 2003).

Reaction mixtures of 100 μL contained 1×PCR buffer, each deoxynucleosidetriphosphate (dNTP) at a concentration of 280 μM, 1.5 mM MgCl₂, 12.5 μMof tetramethylammonium chloride (TMAC), 77 μM of each primer and 10U ofPlatinum Taq (Invitrogen, Madison, Wis.). The high concentration of Taqwas determined by our lab as a means to overcome the effect of minuteamounts of PCR inhibitors. Positive and negative controls were includedto monitor the effects of contaminating DNA found in commercial Taqenzymes. PCR conditions were 95° C. for 5 min, 30 cycles of denaturationat 94° C. for 30 s, annealing at 57.5° C. for 30 s, and extension at 72°C. for 120 s. The final cycle included a final extension at 72° C. for 7min. Purity of PCR products was verified by running in a 1% agarose gel,staining with ethidium bromide and visualizing with a UVtransilluminator. Fluorescently labeled PCR amplicons that wereperformed in triplicate from each sample were pooled and then purifiedfrom the primers and concentrated to 80 μL using a Qiagen PCR Clean UpKit (Qiagen, Valencia, Calif.). Subsequently, the cleaned sample wassplit into four equal volumes. Three of the aliquots were then digestedindividually with 10U of either Bfa I, Hae III, or Msp I individually at37° C. for 4 hr., while the fourth aliquot was stored at −20° C. for afourth restriction enzyme analysis if required. TRFs from digestionsusing Bfa I are denoted with the letter B, while the letter H is used todesignate TRFs from Hae III and M for Msp I. All TRFs designations alsoinclude the size of the fragment, e.g., B 100.79 is a 101 bpTRFgenerated with Bfa I. The use of three restriction enzymes improved thepossibility of taxonomic identification of each TRF to the fewest numberof bacterial species. Digested DNA was then cleaned with a NucleotideClean Up Kit (Qiagen) to improve resolution within the DNA sequencer.Two μL of the T-RFLP product was mixed with 3 μL of premix loadingbuffer that included 2 μL of BlueDextran/EDTA buffer (AppliedBiosystems), 0.5 μL of GeneScan 500 TAMARA size standard (AppliedBiosystems) and 0.5 of formamide. The T-RFs were analyzed byelectrophoresis using a model ABI PRISM 377 Genetic Analyzer (AppliedBiosystems) in Genescan mode (Laragen, Los Angeles, Calif.). GeneScan3.1 software (Applied Biosystems) using the local Southern method wasused to estimate fragment sizes. T-RFs with sizes outside of the rangesof 50-500 by and T-RFs with peak heights below 50 relative fluorescenceunits were removed from the analysis.

Identification of Bacteria by T-RF Matching.

Sample T-RFLP data from each individual pig gut section from eachsampling day was imported into the Bionumerics Gel Compar II packageusing the specialized T-RFLP extension (Applied Maths, Austin, Tex.).The Gel Compar II program was used to facilitate accurate band matchingfor all three restriction enzymes using a 0.5% position tolerance todefine the bacterial species identified as operational taxonomic units(OTU) by T-RFs derived from the three restriction enzymes.

Example 5 Correlation of Gastrointestinal Bacteria to Growth Performanceand Immune Characteristics

Bacteria populations identified in both Examples 1 and 2 were separatelycorrelated to growth performance factors and immune characteristicmeasurements from each trial. Methods for both analyses are described:OTUs were exported into Excel, converted to presence/absence as binarycharacters (0,1) or kept in quantitative form and log₁₀ transformed toprovide a normal distribution. Each data set was plotted and regressedagainst the performance data (ADG, ADFI, pig body weight, and feedefficiency) of each pig using pen as the experimental unit. The immunedata results taken from each individual pig were log₁₀ transformed andregressed against the T-RFLP data of each individual animal at each timepoint. These data were analyzed in two basic ways. First ordinationmethods with graphical plots were applied using Canoco Software Package(v 4.5—Biometris, Wageningen, Netherlands) to understand therelationship of community or population OTU as a whole to that of thestudied parameters (gut section, performance, immune factors ormanagement practice). Second, individual OTUs were regressed againsteach individual variable or parameter to determine direct univariaterelationships. Constrained ordinal methods were applied allowing adetermination of OTU population to variable relationships in thepresence of dominating variables.

Scaling was focused on inter-species correlations and species scoreswere divided by the standard deviation. Sample and species data werecentered but not standardized to avoid overweighting rare species. Forimmunological regressions, all immune factor data was log₁₀ transformedprior to data analyses. Statistical significance of the speciespopulation (all species or OTUs) data in relationship to eachenvironmental variable was determined by Monte Carlo Permutations using499 unrestricted permutations. Statistical significance of eachindividual OTU relating to performance or immune results was alsodetermined in univariate fashion performing General Linear Model(GLM)—Least Squares methodology with the SAS analysis package (SASInstitute, Cary, N.C.) and the Generalized Linear Model with Gaussiandistribution was also applied using Canoco. The latter method is anextension of classical GLM methods. The OTUs with positive or negativeperformance relationships were then putatively identified by comparingagainst the results of all three restriction enzymes using both theMicrobial Community Analysis (MiCA) at the University of Idaho and theT-RFLP Analysis Program (TAP; Marsh et al., 2000) from the RibosomalDatabase Project. The use of three enzymes markedly reduced the numberof potential bacterial species that might be indicative of the specificOTU set and helps to screen out the effect of potential pseudo-TRFs.Using both the population cluster regressions (RDA) and individual OTUregression (GLM) procedures, OTUs were analyzed, independent ofvariables such as treatment or day.

Example 6 Identification of Probiotic Bacteria Based on Correlations toGrowth Performance

Bacteria were selected as potential probiotics based upon significantcorrelations of TRFs to performance criteria, specifically average dailygain, pig body weight, and average daily feed intake. In theconventional vs. segregated early weaning management model described inExample 1, TRFs associated with L. acidophilus were most oftenpositively correlated (P<0.05) to average daily gain during the earlypart of nursery (Phase 1), pig body weight during all three phases ofthe nursery period, and average daily feed intake during the early andlate nursery period (see Table 5 below).

TABLE 5 Correlations associating TRFs identifying specific bacteria togrowth performance measures during the post-weaning period of pigsreared in conventional on-site nursery facilities compared to pigsreared in a segregated early weaning system.¹ Average Daily Gain PigBody Weight Average Daily Feed Intake Phase Phase Phase Phase PhasePhase Phase Phase Phase TRF 1 2a² 2b³ 1 2a² 2b³ 1 2a² 2b³ L. acidophilusB100.79 0.008 0.0001 0.002 0.001 0.014 H330.95 0.005 0.0001 0.002 0.00010.002 0.006 M189.62 0.012 0.003 0.010 0.005 0.028 L. salivarius B262.580.018 0.010 H280.34 (0.008) 0.014 0.024 0.013 0.035 0.038 H279.80 P.acidilactici B274.93 B281.86 0.083 0.026 H337.26 0.080 M582 L.delbruedkii B102.06 0.091 H279.80 M179.95 0.046 0.054 0.045 ¹Valuesdisplayed represent P-values indicating significant (P < 0.10) positive(shown without parentheses) or negative (shown with parentheses)correlations of performance measures and specific TRFs during Phase 1 (d0 to 10 post-weaning) and Phase 2 (d 10 to 24 post-weaning) of thenursery period. ²Phase2a = d 10 to 17 post-weaning. ³Phase2b = d 17 to24 post-weaning.

Other TRFs associated with L. salivarius, P. acidilactici, and L.delbruedkii were also positively correlated (P<0.10) to improved gain,body weight and feed intake. In the model based on pigs reared inconventional confinement farrowing facilities compared to outdoorpasture farrowing facilities, several TRFs associated with L.acidophilus were again positively correlated (P<0.10) to average dailygain, pig body weight, and average daily feed intake (see Table 6below).

TABLE 6 Correlations associating TRFs identifying specific bacteria togrowth performance measures during the post-weaning period of pigsreared in conventional indoor farrowing facilities compared to pigsreared in an outdoor farrowing management system.¹ Day Post-WeaningAverage Daily Gain Pig Body Weight Average Daily Feed Intake TRF d 1 d 3d 10 d 24 d 1 d 3 d 10 d 24 d 1 d 3 d 10 d 24 L. acidophilus B100.66(0.068) 0.059 0.035 (0.073) 0.056 H331.87 (0.065) 0.079 0.071 0.079M189.63 0.087 (0.088) B270.98 (0.090) 0.049 0.089 0.059 0.018 0.0630.027 H336.55 0.059 0.080 0.064 0.085 0.029 0.005 0.029 0.050 H354.76(0.063) 0.006 0.007 0.021 0.019 0.079 0.005 L. salivarius B261.76 0.0780.062 (0.073) 0.074 0.082 0.041 H278.38 M568 P. acidilactici H336.550.059 0.080 0.064 0.085 0.029 0.005 0.029 0.050 B280.97 (0.017) 0.041B274.94 (0.078) M581 L. delbruedkii B102.55 0.066 H278.38 (0.073)M179.75 L. lactis B261.76 0.078 0.062 0.074 0.082 0.041 H278.38 M179.75L. crispatus B265.00 H245.90 (0.063) 0.054 (0.063) M181.86 0.011 (0.096)(0.057) 0.009 ¹Values displayed represent P-values indicatingsignificant (P < 0.10) positive (shown without parentheses) or negative(shown in parentheses) correlations of performance measures and specificTRFs during Phase 1 (d 0 to 10 post-weaning) and Phase 2 (d 10 to 24post-weaning) of the nursery period.

As in Example 1, other TRFs associated with L. salivarius and P.acidilactici were positively correlated (P<0.10) to growth performance.Also, a few TRFs associated with L. delbruedkii, L. lactis, and L.crispatus were positively correlated intermittently with growthperformance factors in the indoor vs. outdoor model. Data associatedwith pre-weaning performance was not collected in the conventional vssegregated early weaning management model. TRFs associated with L.acidophilus, P. acidilactici, and L. crispatus were positivelycorrelated (P<0.10) to piglet body weight during the pre-weaning period,specifically at 6, 13, and 18 days of age in the indoor vs. outdoorrearing model (see Table 7 below).

TABLE 7 Correlations associating TRFs identifying specific bacteria topig body weight during the pre-weaning period of pigs reared inconventional indoor farrowing facilities compared to pigs reared in anoutdoor farrowing management system.¹ Pig Body Weight Days of Age TRF 613 18 L. acidophilus B100.66 0.023 0.049 0.024 H331.87 0.035 0.045 0.046M189.63 B270.98 0.011 0.040 H336.55 0.094 0.042 H354.76 0.001 0.0060.006 P. acidilactici H336.55 0.094 0.042 B280.97 B274.94 M581 L.crispatus B265.00 H245.90 0.003 M181.86 ¹Values displayed representP-values indicating significant (P < 0.10) positive (shown withoutparentheses) or negative (shown in parentheses) correlations ofperformance measures and specific TRFs during Phase 1 (d 0 to 10post-weaning) and Phase 2 (d 10 to 24 post-weaning) of the nurseryperiod.

Correlations to TRFs and day can be used to determine when the bacteriaassociated with specific TRFs should be present in the gastrointestinaltract of the pig, allowing the development of strategies to addresstiming of administration of a probiotic strain. In the conventional vs.segregated early weaning management model, TRFs associated with L.acidophilus were positively correlated (P<0.07) with presence justbefore weaning (18 days of age) and negatively correlated (P<0.05) withpresence early in life (7 days of age) and after weaning (see Table 8below).

TABLE 8 Correlations associating TRFs identifying specific bacteria topresence of the bacteria at specific of pigs reared in conventionalon-site nursery facilities compared to pigs reared in a segregated earlyweaning (SEW) system.¹ Days of age Management System 7 14 18 20 22 30 44Conventional SEW TRF (0.002) (0.0001) L. acidophilus B100.79 0.010(0.0001) (0.058) (0.039) H330.95 (0.0001) (0.0001) (0.026) M189.62(0.001) 0.005 L. salivarius B262.58 0.068 (0.035) (0.011) (0.0001)H280.34 H279.80 0.087 (0.078) (0.030) (0.002) (0.002) (0.0001) (0.0001)P. acidilactici B274.93 B281.86 H337.26 0.001 (0.016) (0.056) 0.0001M582 L. delbruedkii B102.06 H279.80 0.087 (0.078) (0.030) (0.002)(0.002) (0.0001) (0.0001) M179.95 0.017 (0.007) (0.0001) (0.001) (0.039)¹Values displayed represent P-values indicating significant (P < 0.10)positive (shown without parentheses) or negative (shown in parentheses)correlations of age of pig and presence of specific TRFs.

In contrast, TRFs associated with L. delbruedkii were positivelycorrelated (P<0.10) with presence early in life (7 and 14 days of age),but negatively correlated (P<0.10) with presence 18 days of age andlater. Some TRFs associated with L. salivarius and P. acidilactici werepositively and negatively correlated (P<0.10) to presence on variousdays during the pre- and post-weaning periods. In the indoor vs. outdoorrearing model, TRFs associated with L. acidophilus displayed clearnegative correlations with presence before weaning and during the earlyweaning transition (20 days of age) and positive correlations withpresence after 22 days of age (see Table 9 below). Although not asclearly separated between pre- and post-weaning periods, generally TRFsassociated with L. salivarius, L. delbruedkii, L. lactic, and L.crispatus were positively correlated (P<0.05) with presence at 22 daysof age and after, whereas P. acidilactici was positively correlated(P<0.05) with presence pre-weaning and at the weaning transition.

TABLE 9 Correlations associating TRFs identifying specific bacteria tothe presence of the bacteria at specific ages and management systems ofpigs reared in conventional indoor farrowing facilities compared to pigsreared in an outdoor farrowing management system.¹ Days of ageManagement System TRF d 6 13 18 20 22 29 43 Outdoor Indoor L.acidophilus B100.66 (0.023) (0.027) (0.037) (0.013) 0.002 0.0001 0.017H331.87 (0.041) (0.018) 0.045 0.006 (0.087) 0.087 M189.63 (0.083)(0.030) 0.007 0.049 B270.98 (0.094) (0.090) 0.008 0.066 H336.55 (0.057)0.016 0.044 0.020 H354.76 (0.068) (0.064) 0.001 0.058 L. salivariusB261.76 0.024 0.047 (0.047) H278.38 (0.078) 0.022 M568 P. acidilacticiH336.55 (0.057) 0.016 0.044 0.020 B280.97 0.0001 B274.94 (0.053) (0.078)M581 L. delbruedkii B102.55 (0.001) (0.003) 0.027 0.0001 0.037 H278.38(0.078) 0.022 M179.75 L. lactis B261.76 0.02 0.047 (0.047) H278.38M179.75 L. crispatus B265.00 0.058 0.001 (0.015) 0.015 H245.90 (0.004)0.004 M181.86 (0.060) ¹Values displayed represent P-values indicatingsignificant (P < 0.10) positive (shown without parentheses) or negative(shown in parentheses) correlations of age of pig and presence ofspecific TRFs.

Example 7 Identification of Probiotic Bacteria Based on Correlations toImmune Characteristics

Correlations can be made associating specific TRFs with immunepopulations within the systemic circulation (peripheral blood) and thegastrointestinal tract of pigs, allowing the prediction of howadministration of the probiotic bacteria impact immune characteristicsof these tissues in the young pig. Immune populations positively andnegatively associated with specific TRFs from both growth models arelisted in Table 10 and Table 11 below. Generally, TRFs associated withpotential probiotic bacteria correlated positively (P<0.05) toactivated, memory, and gamma-delta T cell subsets in peripheral bloodand the gastrointestinal tract.

TABLE 10 Correlations associating TRFs identifying specific bacteria toimmune cell populations in the peripheral blood and gastrointestinaltract (GIT) of pigs reared in conventional on-site nursery facilitiesand pigs reared in a segregated early weaning (SEW) system.¹ BacterialSpecies TRF Positively Correlated Negatively Correlated L. acidophilusB100.79 1) Blood-Cytotoxic T cells (CD8⁺CD4⁺; 1) Blood-T helper cells(CD3⁺CD4⁺) CD2⁺CD8⁺CD4⁻; CD25⁺CD8⁺CD4⁻) 2) GIT-Activated lymphocytes 2)Blood-Activated memory subset (CD25⁺CD8⁻CD4⁻) (CD25⁺CD8⁺CD4⁺) 3)Blood-Gamma-delta T cells (TCR1⁺CD8⁻CD4⁻) 4) GIT-Memory T cell subset(CD8⁺CD4⁺) H330.95 1) Blood-Gamma-delta T cells (CD8⁻ 1) Blood-Activatedlymphocytes CD4⁻TCR1⁺) (CD25⁺CD8⁻CD4⁻) 2) Blood-Memory T cell subset 2)Blood-Cytotoxic T cells (CD4⁺CD8⁺TCR1⁻) (CD2⁺CD8⁺CD4⁻) M189.62 1)Blood-Memory T cell subset 1) Blood-T lymphocytes (CD3⁺CD4⁻)(CD2⁺CD8⁺CD4⁺) 2) Blood-Gamma-delta T cells 2) GIT-Activated memory Tcell subset (TCR1⁺CD8⁺CD4⁻) (CD25⁺CD8⁺CD4⁺) 3) Blood-Activatedlymphocytes 3) GIT-Gamma-delta T cells (CD25⁺CD8⁻CD4⁻) (CD4⁺CD8⁺TCR1⁺;TCR1⁺CD8⁻CD4⁻) 4) GIT-Cytotoxic T cells (CD8⁺CD4⁻) 5) GIT-Gamma-delta Tcells (TCR1⁺CD8⁺CD4⁺) L. salivarius B262.58 1) Blood-Activatedlymphocytes 1) Blood-Leukocytes with antigen- (CD25⁺CD8⁻CD4⁻) presentingcapacity (MHCII⁺) 2) GIT-Activated T helper cells 2) Blood-T lymphocytes(CD3⁺CD4⁺; (CD4⁺CD25⁺CD8⁻) CD2⁺CD4⁻CD8⁻) 3) GIT-T helper cells(CD2⁺CD4⁺CD8⁻) H279.80 1) Blood-Gamma-delta T cells (TCR⁺; 1)Blood-Leukocytes with antigen- CD4⁺TCR1⁺CD8⁻) presenting capacity(MHCII⁺) 2) GIT-Gamma-delta T cell memory 2) Blood-Activated lymphocytessubset (TCR1⁺CD8⁺CD4⁺) (CD25⁺) 3) Blood-T helper cells (CD3⁺CD4⁺) 4)GIT-T lymphocytes (CD2⁺CD8⁻CD4⁻) 5) GIT-Activated lymphocytes(CD25⁺CD8⁻CD4⁻) 6) GIT-Memory T cell subset (CD8⁺CD4⁺CD25⁻) P.acidilactici B274.93 1) Blood-Gamma-delta T cells 1) Blood-T lymphocytes(CD3⁺CD4⁻; (CD4⁺TCR1⁺CD8⁻) CD2⁺CD4⁻CD8⁻) 2) GIT-Cytotoxic T cells(CD8⁺CD4⁻) 2) Blood-T helper cells (CD4⁺) 3) GIT-Activated lymphocytes3) Blood-Gamma-delta T cells (TCR1⁺) (CD25⁺CD8⁻CD4⁻) 4) Blood-Activatedlymphocytes 4) GIT-Gamma-delta T cells (CD25⁺CD4⁻CD8⁻) (TCR1⁺CD8⁻CD4⁻)5) GIT-T lymphocytes (CD2⁺CD4⁻CD8⁻) 6) GIT-Memory T cell subset(CD4⁺CD8⁺CD2⁺) H337.26 1) Blood-Cytotoxic T cells 1) Blood-Gamma-delta Tcells (CD2⁺CD8⁺CD4⁻) (CD8⁺TCR1⁺CD4⁻; TCR1⁺CD8⁺CD4⁻) 2) Blood-T helpercells (CD3⁺CD4⁺; CD4⁺CD2⁺CD8⁻) ¹Immune cell populations listed arepositively or negatively correlated to the specified TRF at asignificance level of P ≦ 0.05.

TABLE 11 Correlations associating TRFs identifying specific bacteria toimmune cell populations in the peripheral blood and gastrointestinaltract (GIT) of pigs reared in conventional confinement farrowingfacilities and pigs farrowed in an outdoor pasture management system.¹Bacterial Species TRF Positively Correlated Negatively Correlated L.acidophilus B100.66 1) Blood-Activated lymphocytes (CD25⁺CD8⁻CD4⁻;CD25⁺CD8⁺CD4⁻; CD25⁺CD8⁺CD4⁺; CD4⁺CD8⁺CD25⁺; MHCII⁺CD3⁺) 2)Blood-Gamma-Delta T cells (CD4⁺CD8⁺TCR1⁺) 3) Blood-Memory T cell subset(CD8⁺CD4⁺) 4) Blood-B cells (CD21⁺CD25⁻; CD21⁺CD25⁺) 5) GIT-Activatedlymphocytes (CD25⁺CD8⁺CD4⁺; CD4⁺CD8⁺CD25⁺; CD8⁺CD25⁺CD4⁻; CD25⁺CD8⁺CD4⁺;CD4⁺CD25⁺CD8⁻) 6) GIT-Gamma-delta T cells (TCR1⁺CD8⁺CD4⁺; CD4⁺CD8⁺TCR1⁺;CD8⁺TCR1⁺CD4⁻; CD8⁺TCR1⁺CD4⁻; TCR1⁺CD8⁻CD4⁻) 7) GIT-Memory T cellsubsets (CD4⁺CD8⁺TCR1⁻; CD4⁺CD8⁺) H331.87 1) Blood T cells(CD3⁺MHCII⁻) 1) GIT-B cells (CD21⁺CD25⁻) 2) Blood-Activated lymphocytes(CD21⁺CD25⁺; CD25⁺CD21⁻; CD25⁺CD8⁺CD4⁺; CD25⁺CD8⁺CD4⁻; CD25⁺CD8⁻CD4⁺) 3)GIT-Activated T lymphocytes (CD25⁺CD21⁻; CD4⁺CD25⁺CD8⁻; CD25⁺CD4⁺CD8⁻)4) GIT-Memory T cells (CD4⁺CD8⁺CD25⁻; CD8⁺CD4⁺ 5) GIT-Gamma-delta Tcells (CD4⁺TCR1⁺CD8⁻; TCR1⁺CD4⁺CD8⁻) M189.63 1) Blood-T cells(MHCII⁺CD3⁺) 1) GIT-B cells (CD21⁺CD25⁻) 2) Blood-Activated lymphocytes2) GIT-Gamma-delta T cells (CD21⁺CD25⁺; CD25⁺CD21⁻) (TCR1⁺CD8⁺CD4⁻; 3)GIT-T helper cells (CD4⁺) CD4⁺CD8⁺TCR1⁺) 4) GIT-Activated T cells(CD25⁺CD21⁻; CD4⁺CD25⁺CD8⁻; CD25⁺CD8⁻CD4⁻; CD25⁺CD4⁺CD8⁻) 5)GIT-Gamma-delta T cells (CD8⁺TCR1⁺CD4⁻; TCR1⁺CD4⁺CD8⁻ B270.98 1) Blood-Tcells (CD3⁺; MHCII⁺CD3⁺; CD3⁺MHCII⁻; 1) Blood-Activated lymphocytes(CD25⁺CD4⁻ CD3⁺CD8⁻) CD8⁻) 2) Blood-T helper cells (CD4⁺CD8⁻) 3)Blood-Activated lymphocytes (CD21⁺CD25⁺; CD25⁺CD21⁻) 4) GIT-T cells(CD3⁺) 5) GIT-T helper cells (CD4⁺) 6) GIT-Activated lymphocytes(CD25⁺CD8⁻CD4⁻; CD25⁺CD21⁻; CD25⁺CD4⁺CD8⁻) 7) GIT-Gamma-delta T cells(TCR1⁺CD4⁻CD8⁻; H336.55 1) Blood-B and T lymphocytes (CD3⁺; CD3⁺MHCII⁺;CD21⁺CD25⁻) 2) Blood-Activated lymphocytes (CD25⁺; CD25⁺CD8⁺CD4⁺;CD25⁺CD21⁺; CD25⁺CD21⁻; CD8⁺CD25⁺CD4⁻; CD25⁺CD4⁻CD8⁻; CD25⁺CD8⁺CD4⁻) 3)Blood-Gamma-delta T cells (TCR1⁺CD8⁺CD4⁻; TCR1⁺CD4⁻CD8⁻) 4)GIT-Activated lymphocytes (CD25⁺CD21⁻; CD25⁺CD4⁻CD8⁻) 5) GIT-Gamma-deltaT cells (TCR1⁺CD4⁺CD8⁻; H354.76 1) Blood-T cells (CD3⁺MHCII⁻;CD3⁺CD8⁻) 1) Blood-Memory T cell subset (CD4⁺CD8⁺) 2) Blood-Activatedlymphocytes (CD21⁺CD25⁺; CD25⁺CD21⁻; CD3⁺MHCII⁺) 3) GIT-T lymphocytes(CD3⁺) 4) GIT-T helper cells (CD4⁺) 5) GIT-Activated T cells(CD25⁺CD4⁺CD8⁺) 6) GIT-Memory T cell subset (CD4⁺CD8⁺TCR1⁻) L.salivarius B261.76 1) Blood-T cells (MHCII⁺CD3⁺) 1) Blood-Leukocyteswith antigen-presenting 2) GIT-T helper cells (CD4⁺CD8⁻) capacity(MHCII⁺) H278.38 1) Blood-B cells (CD21⁺CD25⁻) 1) Blood-Gamma-delta Tcells 2) Blood-Gamma-delta T cells (TCR1⁺CD8⁻CD4⁻; (TCR1⁺CD4⁺CD8⁻)CD8⁺TCR1⁺CD4⁻) 3) Blood-Activated lymphocytes (CD25⁺CD8⁻CD4⁻) 4)GIT-Gamma-delta T cells (TCR1⁺CD4⁺CD8⁻; TCR1⁺CD8⁺CD4⁺) P. acidilacticiH336.55 See L. acidophilus B280.97 1) GIT-B cells (CD21⁺CD25⁻) 1)Blood-Leukocytes with antigen-presenting 2) GIT-Activated T cells(CD25⁺CD8⁺CD4⁻) capacity (MHCII⁺) 3) GIT-Gamma-delta T cells(TCR1⁺CD8⁺CD4⁻) 2) GIT-Activated T cells (CD25⁺CD4⁺CD8⁻; CD4⁺CD25⁺Cd8⁻)B274.94 1) Blood-T cells (CD3⁺MHCII⁻; CD8⁺CD3⁺; CD4⁺CD8⁻; CD3⁺CD8⁻) 2)Blood-B cells (CD21⁺CD25⁺; CD21⁺) 3) Blood-Gamma-delta T cells(TCR1⁺CD4⁺CD8⁻; 4) Blood-Memory T cell subsets (CD8⁺CD4⁺; CD25⁺CD8⁺CD4⁺)5) GIT-Memory T cell subsets (CD8⁺CD4⁺; CD4⁺CD8⁺TCR1⁻; CD4⁺CD8⁺CD25⁻; 6)GIT-Activated lymphocytes (CD25⁺CD8⁻CD4⁻; CD4⁺CD8⁺CD25⁺; CD25⁺CD4⁺CD8⁻;CD25 + CD4⁺CD8⁻; CD25⁺CD21⁻) 7) GIT-Gamma-delta T cells (CD8⁺TCR1⁺CD4⁻;CD4⁺CD8⁺CD25⁺; CD4⁺CD25⁺CD8⁻; TCR1⁺CD4⁻ CD8⁻) ¹Immune cell populationslisted are positively or negatively correlated to the specified TRF at asignificance level of P ≦ 0.05.

Example 8 Strain Identification of Probiotic Bacteria Through RAPD PCRAnalysis

Intestinal samples that had yielded high peak heights for the TRFs ofinterest were selectively plated for lactic acid producing bacteria.Colonies were picked into broth and grown for 24 hours at which pointDNA was isolated from the cell culture. Similarity of the isolates wasevaluated by comparing RAPD PCR fingerprints of a5′-tetrachlorofluorescein labeled 8F domain primer (5′AGAGTTTGATYMTGGCTCAG 3′) and a 1406R universal primer (5′ACGGGCGGTGTGTRC 3′). TRFLP using Bfa I, Hae III, and Msp I was alsoperformed on the isolates to ensure that they possessed the TRFs ofinterest. The TRFs of interest were all of the TRFs in Tables 5, 6, 7,and 8 that were associated with increased performance. The sizes of theTRFs in Tables 5 and 6 from the On-Site/Off-Site trial and theIndoor/Outdoor trial are slightly different due to position toleranceand optimization settings used in the Bionumerics software but wereconsidered to be the same during the strain selection process. Table 12shows the TRFs of interest with the TRF length and the TRF length plusand minus 2 basepairs to account for the slight differences noted above.The inventors believe that there is about 90% sequence identity whencomparing a TRF sequence from one lactic acid bacteria strain comparedto a TRF sequence from another lactic acid bacteria strain if thestrains have the same TRF.

RAPD PCR fingerprints, TRFLP profiles, and the resulting dendrogramassociated with nine clusters of RAPDs contained one or more TRF ofinterest as shown above are displayed in FIG. 2. Four clusters containedTRFs associated with Lactobacillus acidophilus, three contained TRFsassociated with Lactobacillus salivarius, and two contained TRFsassociated with Pediococcus acidilactici. A representative strain waschosen from each cluster: L. acidophilus o2461L7fv, L. acidophilusP1Bc8, L. acidophilus P1B c6, L. acidophilus PLGf1, L. salivariuso246e8, L. salivarius PL2i4, L. salivarius o246e 33w, P. acidilacticio246e 42, and P. acidilactici P1J e3.

TABLE 12 TRFs of interest determined by correlations associating TRFs toperformance and immunology factors. On-site/ Indoor/ Off-site TRFOutdoor TRF TRF Length from Length from TRF Length Length SpeciesExample 1 Example 2 Rounded Off +/−2 bps L. acidophilus B100.79 B100.66101  99-103 H330.95 H331.87 331-332 329-334 M189.62 M189.63 190 188-192H354.07 H354.76 354-355 352-357 B269.57 B270.98 270-271 268-273 L.salivarius B262.58 B261.76 262-263 260-265 H279.80 H278.38 279-280277-282 P. acidilactici H337.26 H336.55 337 335-339 B281.86 B280.97281-282 279-284 B279.93 280 278-282 B274.93 B274.94 275 273-277

Identification of these nine candidate strains within a dendrogramencompassing the RAPD profiles from all of the lactic acid bacterialisolates reveals that these nine candidate DFM strains have RAPDprofiles that are distinct from each other, even within strains of thesame species. For example, P. acidilactici strain o246e 42 is only ˜10%similar to P. acidilactici strain P1J e3 according to their RAPDprofiles, with many bacterial strains of different species havinggreater similarity to these strains of the same species than they do toeach other.

Example 9 Animal Testing of Probiotic Strains to Determine the Benefitsof Supplementation During the Pre- and Post-Weaning Periods

One strain from each species listed above was selected for initialanimal testing to validate the association with the presence of thesespecific bacteria with improved growth performance in the young pig.Three probiotic strains, including Lactobacillus acidophilus PIB c6,Lactobacillus salivarius o246e 33w, and Pediococcus acidilactici P1J e3,were selected for testing to determine the efficacy of the selectedprobiotic strains for improving growth performance of young pigs duringthe pre- and post-weaning production periods. The three potentialprobiotic strains, Lactobacillus acidophilus PIB c6, Lactobacillussalivarius o246e 33w, and Pediococcus acidilactici P1J e3, were includedin combination in sow diets and individually in nursery pig diets afterweaning. Sows were divided into eight treatment groups of four sows pertreatment, and litters were randomly assigned to one of eight treatmentsto determine the effect of administering the probiotic organisms duringpre- and post-weaning, and in combination or individually during thenursery period (see Table 13 below). Sows (pre-weaning) were topdressed;for nursery pigs (post-weaning), the DFM was mixed into the diet as partof the complete ration. The eight probiotic treatments were formulatedto deliver 1×10⁹ total cfu/pig/day to sows or pigs regardless of whetherthe total cfu was derived from one organism or a combination of thethree selected organisms, with equal amounts (based on CFU5) of eachorganism included where multiple organisms were used.

TABLE 13 Dietary probiotic treatments administered to sows and theirlitters during the lactation¹ and nursery² phases. Treatment LactationNursery 1 Control Control 2 Control L. acidophilus PlB c6 3 Control L.salivarius o246e 33w 4 Control P. acidilactici PlJ e3 5 L. acidophilusPlB c6 Control L. salivarius o246e 33w P. acidilactici PlJ e3 6 L.acidophilus PlB c6 L. acidophilus PlB c6 L. salivarius o246e L.salivarius o246e 33w 33w P. acidilactici PlJ e3 P. acidilactici PlJ e3 7Control L. acidophilus PlB c6 L. salivarius o246e 33w P. acidilacticiPlJ e3  8³ L. acidophilus PlB c6 L. acidophilus PlB c6 L. salivariuso246e L. salivarius o246e 33w 33w P. acidilactici PlJ e3 P. acidilacticiPlJ e3 L. brevis 1E1 L. brevis 1E1 ¹Diets were administered to littersby addition of the probiotic treatment into the sow feed to deliver 1 ×10⁹ cfu/sow/day. ²Probiotic treatments were administered in the nurserypig diet for two weeks following weaning. ³ Lactobacillus brevis wasadded to the three probiotic combo. L. brevis has previously documentedbenefits to the young pig (Davis et al, 2006).

Contrast statements comparing the three treatments with the three straincombination demonstrate the combination of L. acidophilus P1B c6, L.salivarius o246e 33w, and P. acidilactici P1J e3 when administered tothe sow, improves (P=0.03) piglet body weight and average daily gainduring the third week of the lactation period (see Table 14 below). Dueto the limited replication in the nursery period (4replications/treatment), any differences in nursery pig performance thatmay have resulted from the probiotic treatments could not be detected(data not shown).

TABLE 14 Litter performance of pigs nursing sows supplemented with acombination of probiotic strains compared to pigs nursing unsupplementedsows. Lactation L. acidophilus, L. acidophilus, L. acidophilus, L.salivarius, L. salivarius, L. salivarius, P. acidilactici, P valueControl Control Control Control P. acidilactici P. acidilactici ControlL. brevis DFM vs. TRT 1 2 3 4 5 6 7 8 SEM Treatment Control** LitterWeight, lbs. Initial 38.50 38.53 38.73 37.35 38.95 38.78 38.75 38.652.00 0.70 0.35 Period 1 end** 56.63 54.33 51.90 55.42 56.60 57.73 53.4057.53 2.69 0.62 0.11 Period 2 end** 98.88 98.07 93.33 97.53 100.15106.00 94.78 101.42 6.20 0.81 0.15 Period 3 end** 142.85 132.25 135.00138.75 142.38 150.95 136.25 143.50 8.55 0.77 0.15 Litter Size, pigs/sowInitial 11.00 11.00 11.00 11.00 11.00 11.00 11.00 11.00 0.00 1.00 1.00Period 1 end 11.00 10.25 10.25 11.00 10.75 10.50 10.75 11.00 0.32 0.400.66 Period 2 end 10.75 10.25 10.00 10.50 10.25 10.50 10.50 10.50 0.460.96 0.96 Period 3 end 10.75 9.50 9.75 10.50 10.00 10.25 10.50 10.000.40 0.39 0.70 Ave. Pig wt., lbs Initial 3.50 3.50 3.52 3.39 3.54 3.533.52 3.52 0.18 0.69 0.33 Period 1 end 5.15 5.31 5.05 5.04 5.28 5.53 4.955.23 0.22 0.45 0.08 Period 2 end 9.19 9.60 9.32 9.26 9.82 10.16 9.009.64 0.47 0.52 0.06 Period 3 end 13.29 13.95 13.83 13.20 14.34 14.8312.92 14.36 0.75 0.33 0.03 Body wt gain, lbs/d Period 1 0.38 0.40 0.340.36 0.39 0.45 0.32 0.39 0.04 0.37 0.09 Period 2 0.58 0.61 0.61 0.600.65 0.66 0.58 0.63 0.04 0.71 0.08 Period 3 0.59 0.62 0.64 0.56 0.650.67 0.56 0.68 0.05 0.13 0.02 Cumulative 0.53 0.57 0.56 0.53 0.59 0.610.51 0.59 0.04 0.34 0.03 *Values represent the mean of four litters pertreatment **Contrast of Treatments 1, 2, 3, 4, and 7 vs. Treatments 5,6, and 8.

Example 10 Animal Testing to Confirm and Further Define the BeneficialResponse from Supplementation of a Combination of Three ProbioticStrains to Sows During Lactation

The beneficial response from feeding the three-strain probioticcombination of Lactobacillus acidophilus P1B c6, Lactobacillussalivarius o246e 33w, and Pediococcus acidilactici P1J e3 to sows duringthe lactation period was confirmed in a second study that furtherevaluated these strains by also testing P. acidilactici P1J e3 and L.salivarius o246e 33w in combination as well as P. acidilactici P1J e3alone in the following treatment arrangement with 20 pens per treatment:(1) A control diet, (2) A control diet supplemented with all threedirect fed microbial strains at a total count of 1×10⁹ total cfu/pig/dayincluding 3.34×10⁸ cfu/pig/day each of Pediococcus acidilactici P1J e3,Lactobacillus salivarius o246e 33w, and Lactobacillus acidophilus PIBc6, (3) A control diet supplemented with two of the direct fed microbialstrains at a total count of 1×10⁹ including 5.0×10⁸ each of Pediococcusacidilactici P1J e3 and Lactobacillus salivarius o246e 33w, and (4) Acontrol diet supplemented with 1×10⁹ of only Pediococcus acidilacticiP1J e3. All treatments were top dressed to sows.

Supplementation with the three strain combination again improved(P<0.05) piglet average daily gain during the third week of lactationcompared to pigs from unsupplemented sows (see Table 15 below). Althoughnot significantly different from the control, average daily gain ofpiglets nursing sows supplemented with only P. acidilactici P1J e3 wasnumerically greater, and was similar to piglets nursing sowssupplemented with the three strain combination. Supplementation of sowswith either the three strain combination or P. acidilactici PhJ e3 aloneprevented an increase in the variation in piglet weight within a litter,and P. acidilactici P1J e3 supplementation alone decreased the variationin piglet weight within litter throughout the lactation period(treatment×time interaction, P=0.06; see FIG. 3).

TABLE 15 Pre-weaning litter performance of litters from sowsadministered probiotic supplements throughout lactation. Treatments¹Control PA + LS + LA PA + LS PA SE P= Initial Weights at Birth² LitterWeight 36.58 37.00 37.45 37.14 0.99 0.845 Average Pig Weight 3.24 3.283.32 3.29 0.08 0.837 Period 1 ADG 0.37 0.39 0.36 0.39 0.02 0.562 LitterWeight 61.17 60.18 60.04 60.44 1.97 0.964 Average Pig Weight 5.49 5.705.55 5.72 0.16 0.414 Period 2 ADG 0.50 0.54 0.50 0.52 0.022 0.233 LitterWeight 97.97 97.60 95.96 96.50 3.16 0.954 Average Pig Weight 9.26 9.699.20 9.75 0.276 0.143 Period 3 ADG 0.51^(b) 0.56^(a) 0.50^(b) 0.54^(a,b)0.019 0.027 Litter Wean Weight 134.39 135.94 131.22 132.26 4.47 0.807Average Pig Weight ¹PA = Pediococcus acidilactici PlJ e3; LS =Lactobacillus salivarius o246e 33w; LA = Lactobacillus acidophilus PlBc6. ²Mean weights after number of pigs per litter were equalized.^(a,b)Means without common superscripts are significantly different at P< 0.05.

Example 11 Animal Testing of Probiotic Strains to Determine the Benefitsof Supplementation During the Nursery Phase of Production

A total of 480 pigs were weaned, blocked based on initial body weight,and housed four pigs/pen in a total of 120 pens. Six dietary treatmentswere administered to the nursery pigs during the first two weeks of thenursery period (20 pens/treatment). Dietary treatments were 1) A controldiet administered to weanling pigs during the first two weeks of thenursery phase; 2) The control diet supplemented with direct-fedmicrobial candidate organism Lactobacillus acidophilus, P1B c6, at 1×10⁹cfu/pig/day and administered for two weeks in the nursery diet; 3) Thecontrol diet supplemented with direct-fed microbial candidate organismLactobacillus salivarius, o246e 8, at 1×10⁹ cfu/pig/day and administeredfor two weeks in the nursery diet; 4) The control diet supplemented withdirect-fed microbial candidate organism Lactobacillus salivarius, P12i4, at 1×10⁹ cfu/pig/day and administered for two weeks in the nurserydiet; 5) The control diet supplemented with direct-fed microbialcandidate organism Lactobacillus salivarius, o246e 33w, at 1×10⁹cfu/pig/day and administered for two weeks in the nursery diet; 6) Thecontrol diet supplemented with direct-fed microbial candidate organismPediococcus acidilactici, o246e 42, at 1×10⁹ cfu/pig/day andadministered for two weeks in the nursery diet.

Common nursery diets were fed to all pigs for the last four weeks of thetrial. Pig body weight and feed disappearance was determined weekly, andADG, ADFI, and feed efficiency were calculated for each pen during thesix week trial.

Supplementation with L. salivarius o246e 33w improved (p<0.05) feedefficiency during the second week of weaning compared to theunsupplemented pigs and pigs fed L. salivarius P12 i4 (Table 15).Although not statistically significant (P>0.05) from the controltreatment, pigs fed P. acidilactici 0246e 42 had the lowest FE duringthe sixth week of the trial. This same strain and L. salivarius 0246e 8resulted in improved (P<0.05) feed efficiency compared to pigs fed L.acidophilus PIB c6 and L. salivarius P12 i4 (Table 16).

TABLE 16 Growth Performance ¹ Dietary Treatments² 1 2 3 4 5 6 SEM³P-value Control PlB c6 o246e 8 Pl2 i4 o246i 33w o246e 42 Start weight14.21^(b) 14.28^(ab) 14.25^(ab) 14.19^(b) 14.20^(b) 14.35^(a) 0.35 0.036Week 1 weight 16.86 16.74 16.59 16.85 16.73 16.90 0.38 0.866 ADG 0.330.31 0.29 0.33 0.32 0.32 0.02 0.808 ADFI 0.38 0.36 0.36 0.38 0.37 0.370.02 0.937 FE⁴ 1.27 1.28 1.30 1.22 1.22 1.23 0.08 0.928 Week 2 weight21.34 21.14 20.92 20.94 21.25 21.55 0.51 0.660 ADG 0.73 0.73 0.72 0.680.74 0.78 0.04 0.223 ADFI 0.76 0.74 0.73 0.74 0.73 0.79 0.03 0.580 FE1.07^(ab) 1.02^(bc) 1.02^(bc) 1.10^(a) 1.00^(c) 1.02^(bc) 0.03 0.004Week 3 weight 27.33 27.29 27.06 26.99 27.17 27.75 0.63 0.880 ADG 0.740.77 0.76 0.76 0.74 0.77 0.03 0.937 ADFI 1.05 1.05 1.05 1.02 1.04 1.090.04 0.768 FE 1.44 1.39 1.41 1.36 1.41 1.43 0.04 0.582 Week 4 weight34.48 34.81 34.06 34.71 34.67 34.99 0.89 0.880 ADG 1.02 1.08 1.00 1.101.05 1.04 0.07 0.414 ADFI 1.47 1.49 1.45 1.52 1.49 1.48 0.07 0.854 FE1.53 1.40 1.58 1.44 1.48 1.61 0.09 0.207 Week 5 weight 44.01 44.55 43.4644.41 43.74 44.76 1.11 0.783 ADG 1.36 1.39 1.32 1.37 1.30 1.39 0.050.562 ADFI 1.91 1.97 1.82 1.93 1.85 1.95 0.08 0.296 FE 1.41 1.42 1.371.42 1.43 1.40 0.03 0.465 Week 6 weight 53.56 54.08 53.10 53.93 53.3954.79 1.27 0.804 ADG 1.37 1.36 1.38 1.36 1.38 1.43 0.06 0.888 ADFI 2.152.21 2.11 2.20 2.12 2.18 0.08 0.916 FE 1.59^(ab) 1.63^(a) 1.54^(b)1.65^(a) 1.56^(ab) 1.53^(b) 0.04 0.049 Cumulative ADG 0.90 0.93 0.890.92 0.90 0.94 0.03 0.591 ADFI 1.26 1.29 1.23 1.28 1.25 1.30 0.04 0.562FE 1.40 1.39 1.38 1.39 1.38 1.38 0.01 0.551 ¹ Data are means of 20replicates of six treatments. ²Dietary treatments were treatment 1 =Control, treatment 2 = control diet supplemented with direct-fedmicrobial candidate organism Lactobacillus acidophilus, PlB c6, at 1 ×10⁹ cfu/pig/day, treatment 3 = control diet supplemented with direct-fedmicrobial candidate organism Lactobacillus salivarius, o246e 8, at 1 ×10⁹ cfu/pig/day, treatment 4 = control diet supplemented with direct-fedmicrobial candidate organism Lactobacillus salivarius, Pl2 i4, at 1 ×10⁹ cfu/pig/day, treatment 5 = control diet supplemented with direct-fedmicrobial candidate organism Lactobacillus salivarius, o246i 33w, at 1 ×10⁹ cfu/pig/day, treatment 6 = control diet supplemented with direct-fedmicrobial candidate organism Pediococcus acidilactici, o246e 42, at 1 ×10⁹ cfu/pig/day ³standard error of the mean (SEM) ⁴ feed efficiency (FE)^(abc)Means within a row with different superscripts are significantlydifferent (P < 0.05)

Example 12 Identification of Probiotic Bacteria Based on NegativeCorrelations to Potentially Pathogenic Bacteria

Correlations can be made associating presence of specific TRFs in thegastrointestinal tract representing selected probiotic strains with thepresence of pathogen defined TRFs, allowing the prediction of howadministration of the probiotic bacteria impact the presence ofpathogenic organisms in these tissues in the young pig. The presence ofTRFs defined as L. acidophilus (Table 17) and L. salivarius (Table 18)correlated negatively (P<0.05) to the presence of severalpathogen-defined TRFs defined as Clostridium, Mycobacterium, andPasteurella spp indicating that when these probiotic bacteria werepresent in the gastrointestinal tract, these pathogens were less likelyto be present.

TABLE 17 Terminal restriction fragments (TRFs) that were negativelycorrelated to the presence of L. acidophilus-defined TRFs (B100.79,H330.95, M189.62) in the gastrointestinal tracts of pigs. L. acidophilusCorrelation Putative TRF P value Identification B100.79 H231.09 0.034Clostridium spp., Mycobacterium spp. H180.00 0.012 Mycobacterium spp.H330.95 H231.09 0.001 Clostridium spp., Mycobacterium spp. M281.97 0.003Mycobacterium spp. M474.33 0.011 Clostridium spp. H256.70 0.021Pasteurella spp. H258.50 0.047 Clostridium spp. M189.62 B279.93 0.027Pasteurella spp.

TABLE 18 Terminal restriction fragments (TRFs) that were negativelycorrelated to the presence of L. salivarius-defined TRFs in thegastrointestinal tracts of pigs. L. salivarius Correlation TRF P valuePutative Identification B262.58 H231.09 0.016 Clostridium spp.,Mycobacterium spp. M281.97 0.016 Mycobacterium spp. H180.00 0.018Mycobacterium spp. B336.73 0.034 Clostridium spp. M474.33 0.037Clostridium spp. M71.77 0.048 Mycobacterium spp. B55.08 0.008Clostridium spp. H301.08 0.004 Clostridium spp. H279.80 M474.33 0.050Clostridium spp.

It is understood that the various embodiments are shown and describedabove to illustrate different possible features of the invention and thevarying ways in which these features may be combined. Apart fromcombining the different features of the above embodiments in varyingways, other modifications are also considered to be within the scope ofthe invention. The invention is not intended to be limited to theembodiments described above, but rather is intended to be limited onlyby the claims set out below. Thus, the invention encompasses allalternate embodiments that fall literally or equivalently within thescope of these claims.

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What is claimed is:
 1. A DNA sequence including a portion of the 16S rRNA gene coding region of a lactic acid bacteria, the 5′ end of which includes the sequence of 5′ AGAGTTTGATYMTGGCTCAG 3′, the 3′ end of which includes a restriction enzyme recognition site for one of Bfa I, Hae III, and Msp I, when the restriction enzyme recognition site is Bfa I, the DNA sequence having a length of about 99 base pairs to about 103 base pairs, about 268 base pairs to about 273 base pairs, about 260 base pairs to about 265 base pairs, about 279 base pairs to about 284 base pairs, about 278 base pairs to about 282 base pairs, or about 273 base pairs to about 277 base pairs, when the restriction enzyme recognition site is Hae III, the DNA sequence having a length of about 329 base pairs to about 334 base pairs, about 352 base pairs to about 357 base pairs, about 277 base pairs to about 282 base pairs, or about 335 base pairs to about 339 base pairs, and when the restriction enzyme recognition site is Msp I, the DNA sequence having a length of about 188 base pairs to about 192 base pairs.
 2. A method of identifying one or more strain that can be used as a direct-fed microbial, the method comprising: isolating DNA from bacteria from the gastrointestinal tract of an animal; amplifying the DNA; analyzing the amplified DNA with T-RFLP to generate TRF data; correlating the TRF data to a characteristic of interest; identifying strains of interest from the correlations; and; confirming the presence of the TRF in the strain.
 3. A strain identified by the method of claim
 2. 4. The strain of claim 3, wherein the strain is a potential probiotic.
 5. The strain of claim 3, wherein the strain is a potential pathogen.
 6. A method comprising administering to an animal an effective amount of at least one strain of claim
 3. 7. An isolated Pediococcus acidilactici strain P1J e3 (NRRL B-50101).
 8. A combination comprising: the strain of claim 7; and an isolated Lactobacillus salivarius strain o246e 33w (NRRL B-50102).
 9. The combination of claim 8, further comprising an isolated Lactobacillus acidophilus strain PIB c6 (NRRL B-50103).
 10. An isolated strain chosen from at least one of Lactobacillus acidophilus strain PIB c6 (NRRL B-50103), Lactobacillus salivarius strain o246e 33w (NRRL B-50102), and Pediococcus acidilactici strain o246e 42 (NRRL B-50171).
 11. An isolated strain having all of the identifying characteristics of one of the strains of claim
 10. 12. The strain of claim 10, further comprising a carrier.
 13. A method comprising administering to an animal an effective amount of at least one strain chosen from Lactobacillus acidophilus strain P1B c6 (NRRL B-50103), Lactobacillus salivarius strain o246e 33w (NRRL B-50102), Pediococcus acidilactici strain o246e 42 (NRRL B-50171), and Pediococcus acidilactici strain P1J e3 (NRRL B-50101).
 14. The method of claim 13, wherein the strain is Pediococcus acidilactici strain P1J e3 (NRRL B-50101).
 15. The method of claim 13, wherein the strains are Pediococcus acidilactici strain P1J e3 (NRRL B-50101) and Lactobacillus salivarius strain o246e 33w (NRRL B-50102).
 16. The method of claim 13, wherein the strains are Pediococcus acidilactici strain P1J e3 (NRRL B-50101), Lactobacillus salivarius strain o246e 33w (NRRL B-50102), and Lactobacillus acidophilus strain P1B c6 (NRRL B-50103).
 17. The method of claim 13, wherein the animal is a pig.
 18. The method of claim 17, wherein about 1×10⁸ total cfu/pig/day to about 5×10¹⁰ total cfu/pig/day of the strain or strains is administered to the pig.
 19. The method of claim 13, wherein the animal is a sow.
 20. The method of claim 19, wherein the sow is a lactating sow.
 21. The method of claim 19, wherein the strains are Pediococcus acidilactici strain P1J e3 (NRRL B-50101), Lactobacillus salivarius strain o246e 33w (NRRL B-50102), and Lactobacillus acidophilus strain P1B c6 (NRRL B-50103); and further comprising improving body weight and average daily gain in piglets borne to the sow relative to that in piglets borne to sows that have not been administered the strains.
 22. The method of claim 19, wherein the strains are Pediococcus acidilactici strain P1J e3 (NRRL B-50101), Lactobacillus salivarius strain o246e 33w (NRRL B-50102), and Lactobacillus acidophilus strain P1B c6 (NRRL B-50103); and further comprising preventing an increase in variation in piglet weight within a litter borne to the sow relative to that in piglets borne to sows that have not been administered the strains.
 23. The method of claim 19, wherein the strain is Pediococcus acidilactici strain P1J e3 (NRRL B-50101); and further comprising preventing an increase in variation in piglet weight within a litter borne to the sow relative to that in piglets borne to sows that have not been administered the strains.
 24. The method of claim 19, wherein the strain is Pediococcus acidilactici strain P1J e3 (NRRL B-50101); and further comprising decreasing variation in piglet weight within a litter borne to the sow relative to that in piglets borne to sows that have not been administered the strains.
 25. The method of claim 13, wherein the animal is a pig in the nursery phase of production, and wherein the strain is Pediococcus acidilactici strain o246e 42 (NRRL B-50171).
 26. The method of claim 13, further comprising modulating the immune system of the animal with the strain.
 27. A method of preparing a direct-fed microbial, the method comprising: (a) growing, in a liquid nutrient broth, at least one strain chosen from Lactobacillus acidophilus strain P1B c6 (NRRL B-50103), Lactobacillus salivarius strain o246e 33w (NRRL B-50102), Pediococcus acidilactici strain o246e 42 (NRRL B-50171), and Pediococcus acidilactici strain P1J e3 (NRRL B-50101); and (b) separating the strain from the liquid nutrient broth to form the direct-fed microbial.
 28. The method of claim 27, wherein the strain is Pediococcus acidilactici strain P1J e3 B-50101. 